PROJECT SUMMARY/ABSTRACT Allergenic proteins belong to a relatively small proportion of protein families. Our analysis revealed that only 1.4% (130 out of 9,316) of the protein family database (Pfam) contain allergens. Pectate lyase allergens reside in a single protein family Pfam code PF00544. Hypersensitivity to pectate lyase allergens, e.g., the major allergens of ragweed and cedar pollens, causes severe seasonal allergic diseases in highly populated regions of the world? including the US, South America, Europe and Asia. The National Health and Nutrition Examination Survey (NHANES) indicated that 44.6% of the US population (age > 6 years) was sensitized to one or more allergens, and that grasses, dust mites and ragweed are the most common allergens. Other studies indicate that about 30% of the US population is sensitized to ragweed. We have extensively characterized one of the pectate lyase allergens, Jun a 1 from mountain cedar (Juniperus ashei) pollen, including its crystal structure and identification of its epitopes to use as our model for this study. The structures of Jun a 1, and Amb a 1 from short ragweed (Ambrosia artemisiifolia) are primarily parallel ?- helices. IgE antibodies from mountain cedar allergic patients predominantly recognize conformational epitopes of Jun a 1. We have found that one of our monoclonal antibodies (mAbs), termed E58, which binds to a linear epitope on Jun a 1, have unique characteristics. Binding of E58 to Jun a 1 significantly reduces the subsequent binding of our four independent groups of mAbs, and importantly, patient serum IgE, and the degranulation of mast cells, that were passively sensitized with patient serum IgE. E58 also inhibits the binding of patient?s IgE to ragweed allergen. These findings suggest that E58 binding to Jun a 1 and Amb a 1 causes modification of their surface structure, and potentially their general molecular structure. Thus, a single inhibitory agent may prevent IgE binding to allergens across one Pfam group, and potentially other closely related protein groups. We hypothesize that identification of the E58 epitopes on these allergens and their E58 complementarity determining regions will provide important information about the molecular mechanism(s), by which antibodies bind to pectate lyase allergens. The investigative approach we will develop for this project may be useful in identifying unique regions on other allergens, which might induce similar surface alteration that can regulate their allergenicity. Our Aims are 1) to identify the Jun a 1 epitope for binding of mAb E58, using synthetic overlapping peptide arrays, and use the optimized sequences to design variants of E58 single chain antibody fragment (scAb) with enhanced affinity, which inhibit binding of pathologic IgE antibodies to each pectate lyase allergen, and 2) to validate the efficacy of these new recombinant E58 scAb variants in preventing the activation of innate and acquired immunity of patients with hypersensitivity to these allergens. These rE58 scAb and/or their variants have the potential to become new human therapeutics. The identified E58 and/or its variant epitope peptides could also become vaccines for cedar and ragweed pollen hypersensitivity.